Cytokinin-responsive MdTCP17 interacts with MdWOX11 to repress adventitious root primordium formation in apple rootstocks.

Adventitious root (AR) formation plays an important role in vegetatively propagated plants. Cytokinin (CK) inhibits AR formation, but the molecular mechanisms driving this process remain unknown. In this study, we confirmed that CK content is related to AR formation and further revealed that a high auxin/CK ratio was beneficial to AR formation in apple (Malus domestica). A correlation between expression of CK-responsive TEOSINTE BRANCHED1, CYCLOIDEA, and PCF17 (MdTCP17) and AR formation in response to CK was identified, and overexpression of MdTCP17 in transgenic apple inhibited AR formation. Yeast two-hybrid, bimolecular fluorescence complementation, and co-immunoprecipitation assays revealed an interaction between MdTCP17 and WUSCHEL-RELATED HOMEOBOX11 (MdWOX11), and a significant correlation between the expression of MdWOX11 and AR ability. Overexpression of MdWOX11 promoted AR primordium formation in apple, while interference of MdWOX11 inhibited AR primordium production. Moreover, a positive correlation was found between MdWOX11 and LATERAL ORGAN BOUNDARIES DOMAIN29 (MdLBD29) expression, and yeast one-hybrid, dual luciferase reporter, and ChIP-qPCR assays verified the binding of MdWOX11 to the MdLBD29 promoter with a WOX-box element in the binding sequence. Furthermore, MdTCP17 reduced the binding of MdWOX11 and MdLBD29 promoters, and coexpression of MdTCP17 and MdWOX11 reduced MdLBD29 expression. Together, these results explain the function and molecular mechanism of MdTCP17-mediated CK inhibition of AR primordium formation, which could be used to improve apple rootstocks genetically.

Methylation of a MITE insertion in the MdRFNR1-1 promoter is positively associated with its allelic expression in apple in response to drought stress.

Miniature inverted-repeat transposable elements (MITEs) are widely distributed in the plant genome and can be methylated. However, whether DNA methylation of MITEs is associated with induced allelic expression and drought tolerance is unclear. Here, we identified the drought-inducible MdRFNR1 (root-type ferredoxin-NADP+ oxidoreductase) gene in apple (Malus domestica). MdRFNR1 plays a positive role in drought tolerance by regulating the redox system, including increasing NADP+ accumulation and catalase and peroxidase activities and decreasing NADPH levels. Sequence analysis identified a MITE insertion (MITE-MdRF1) in the promoter of MdRFNR1-1 but not the MdRFNR1-2 allele. MdRFNR1-1 but not MdRFNR1-2 expression was significantly induced by drought stress, which was positively associated with the MITE-MdRF1 insertion and its DNA methylation. The methylated MITE-MdRF1 is recognized by the transcriptional anti-silencing factors MdSUVH1 and MdSUVH3, which recruit the DNAJ domain-containing proteins MdDNAJ1, MdDNAJ2, and MdDNAJ5, thereby activating MdRFNR1-1 expression under drought stress. Finally, we showed that MdSUVH1 and MdDNAJ1 are positive regulators of drought tolerance. These findings illustrate the molecular roles of methylated MITE-MdRF1 (which is recognized by the MdSUVH-MdDNAJ complex) in induced MdRFNR1-1 expression as well as the drought response of apple and shed light on the molecular mechanisms of natural variation in perennial trees.

The chromatin remodeller MdRAD5B enhances drought tolerance by coupling MdLHP1-mediated H3K27me3 in apple.

RAD5B belongs to the Rad5/16-like group of the SNF2 family, which often functions in chromatin remodelling. However, whether RAD5B is involved in chromatin remodelling, histone modification, and drought stress tolerance is largely unclear. We identified a drought-inducible chromatin remodeler, MdRAD5B, which positively regulates apple drought tolerance. Transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) analysis showed that MdRAD5B affects the expression of 466 drought-responsive genes through its chromatin remodelling function in response to drought stress. In addition, MdRAD5B interacts with and degrades MdLHP1, a crucial regulator of histone H3 trimethylation at K27 (H3K27me3), through the ubiquitin-independent 20S proteasome. Chromatin immunoprecipitation-sequencing (ChIP-seq) analysis revealed that MdRAD5B modulates the H3K27me3 deposition of 615 genes in response to drought stress. Genetic interaction analysis showed that MdRAD5B mediates the H3K27me3 deposition of drought-responsive genes through MdLHP1, which causes their expression changes under drought stress. Our results unravelled a dual function of MdRAD5B in gene expression modulation in apple in response to drought, that is, via the regulation of chromatin remodelling and H3K27me3.

The SET-Domain-Containing Protein MdSDG26 Negatively Regulates Alternaria alternata Resistance in Apple.

Apple leaf spot is one of the most devastating diseases in the apple industry, caused by Alternaria alternata f. sp mali (A. alternata). SET-domain group (SDG) proteins function as the histone methyltransferases and participate in plant development and stress responses. However, whether SDG proteins are associated with A. alternata resistance is largely unclear. Here, we describe the pathogen-inducible MdSDG26 gene in apple (Malus × domestica). MdSDG26 has two transcript variants that function similarly in catalyzing histone methylation and A. alternata resistance. Transient overexpression of MdSDG26 increased the global levels of H3K4me3 and H3K36me3, whereas knockdown of MdSDG26 only reduced the H3K36me3 level. Transcriptome analysis revealed that MdSDG26 affected the genome-wide transcriptome changes in response to A. alternata infection. ChIP-qPCR analysis demonstrated that MdSDG26 modulates the levels of H3K36me3 and H3K4me3 at both the promoter and exon regions of MdNTL9. As a negative regulator of A. alternata resistance in apples, MdNTL9 plays a pivotal role in MdSDG26-mediated resistance to A. alternata. Therefore, our findings provide compelling evidence for the regulatory function of MdSDG26 in histone methylation and its molecular role in conferring resistance to A. alternata.

MdGH3.6 is targeted by MdMYB94 and plays a negative role in apple water-deficit stress tolerance.

Drought significantly limits apple fruit production and quality. Decoding the key genes involved in drought stress tolerance is important for breeding varieties with improved drought resistance. Here, we identified GRETCHEN HAGEN3.6 (GH3.6), an indole-3-acetic acid (IAA) conjugating enzyme, to be a negative regulator of water-deficit stress tolerance in apple. Overexpressing MdGH3.6 reduced IAA content, adventitious root number, root length and water-deficit stress tolerance, whereas knocking down MdGH3.6 and its close paralogs increased IAA content, adventitious root number, root length and water-deficit stress tolerance. Moreover, MdGH3.6 negatively regulated the expression of wax biosynthetic genes under water-deficit stress and thus negatively regulated cuticular wax content. Additionally, MdGH3.6 negatively regulated reactive oxygen species scavengers, including antioxidant enzymes and metabolites involved in the phenylpropanoid and flavonoid pathway in response to water-deficit stress. Further study revealed that the homolog of transcription factor AtMYB94, rather than AtMYB96, could bind to the MdGH3.6 promoter and negatively regulated its expression under water-deficit stress conditions in apple. Overall, our results identify a candidate gene for the improvement of drought resistance in fruit trees.

MdZAT5 regulates drought tolerance via mediating accumulation of drought-responsive miRNAs and mRNAs in apple.

Drought limits apple yield and fruit quality. However, the molecular mechanism of apple in response to drought is not well known. Here, we report a Cys2/His2 (C2H2)-type zinc-finger protein, MdZAT5, that positively regulates apple drought tolerance by regulating drought-responsive RNAs and microRNAs (miRNAs). DNA affinity purification and sequencing and yeast-one hybrid analysis identified the binding motifs of MdZAT5, T/ACACT/AC/A/G. Chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR) and electrophoretic mobility shift assays (EMSAs) showed that MdZAT5 directly binds to the promoters of the drought-responsive genes including MdRHA2a, MdLEA14, MdTPX1, and MdCAT3, and activates their expression under drought stress. MdZAT5 interacts with and directly targets HYPONASTIC LEAVES1 (MdHYL1). MdZAT5 may facilitate the interaction of MdHYL1 with pri-miRNAs or MdDCL1 by activating MdHYL1 expression, thereby regulating the biogenesis of drought-responsive miRNAs. Genetic dissection showed that MdHYL1 is essential for MdZAT5-mediated drought tolerance and miRNA biogenesis. In addition, ChIP-qPCR and EMSA revealed that MdZAT5 binds directly to the promoters of some MIR genes including Mdm-miR171i and Mdm-miR172c, and modulates their transcription. Taken together, our findings improve our understanding of the molecular mechanisms of drought response in apple and provide a candidate gene for the breeding of drought-tolerant cultivars.

Insights into the effect of human civilization on Malus evolution and domestication.

The evolutionary history of the Malus genus has not been well studied. In the current study, we presented genetic evidence on the origin of the Malus genus based on genome sequencing of 297 Malus accessions, revealing the genetic relationship between wild species and cultivated apples. Our results demonstrated that North American and East Asian wild species are closer to the outgroup (pear) than Central Asian species, and hybrid species including natural (separated before the Pleistocene, about 2.5 Mya) and artificial hybrids (including ornamental trees and rootstocks) are between East and Central Asian wild species. Introgressions from M. sylvestris in cultivated apples appeared to be more extensive than those from M. sieversii, whose genetic background flowed westward across Eurasia and eastward to wild species including M. prunifolia, M. × asiatica, M. × micromalus, and M. × robust. Our results suggested that the loss of ancestral gene flow from M. sieversii in cultivated apples accompanied the movement of European traders around the world since the Age of Discovery. Natural SNP variations showed that cultivated apples had higher nucleotide diversity than wild species and more unique SNPs than other apple groups. An apple ERECTA-like gene that underwent selection during domestication on 15th chromosome was identified as a likely major determinant of fruit length and diameter, and an NB-ARC domain-containing gene was found to strongly affect anthocyanin accumulation using a genome-wide association approach. Our results provide new insights into the origin and domestication of apples and will be useful in new breeding programmes and efforts to increase fruit crop productivity.

Abscisic acid homeostasis is mediated by feedback regulation of MdMYB88 and MdMYB124.

The phytohormone abscisic acid (ABA) is involved in various plant processes. In response to drought stress, plants quickly accumulate ABA, but the regulatory mechanism of ABA accumulation is largely unknown, especially in woody plants. In this study, we report that MdMYB88 and MdMYB124 are myeloblastosis (MYB) transcription factors critical for ABA accumulation in apple trees (Malus x domestica) following drought, and this regulation is negatively controlled by ABA. MdMYB88 and MdMYB124 positively regulate leaf water transpiration, photosynthetic capacity, and stress endurance in apple trees under drought conditions. MdMYB88 and MdMYB124 regulate the expression of biosynthetic and catabolic genes of ABA, as well as drought- and ABA- responsive genes. MdMYB88 associates with promoter regions of the ABA biosynthetic gene 9-cis-epoxycarotenoid dioxygenase 3 (NCED3). Finally, expression of MdMYB88 and MdMYB124 is repressed by ABA. Our results identify a feedback regulation of MdMYB88 and MdMYB124 in modulating ABA homeostasis in apple trees.

Molecular characterizations and expression profiles of transient receptor potential channels in the brown planthopper, Nilaparvata lugens.

Transient receptor potential (TRP) is a superfamily of important cation channels located on the cell membrane. It can regulate almost all sensory modality and control a series of behaviors, including hearing, locomotion, gentle touch, temperature sensation, dry air and food texture detection. The expression profiles of TRP channels have been well documented in the model insect Drosophila melanogaster. However, little is known about the TRP channels of agricultural pests. In this study, we cloned 9 TRP ion channel genes from brown planthopper. Their amino acid sequences are highly conserved with homologues of other insects and have typical TRP channel characteristics: six transmembrane domains (TM1 - TM6) and a pore region between TM5 and TM6. These TRP channels of N. lugens were expressed in all developmental stages and various body parts. The expression levels of almost all TRP channels were relatively higher in adults than nymph stages, and lowest in the eggs. Antenna and abdomen were the main body parts with high expression of these genes. Furthermore, the mRNA levels of these TRP genes were significantly decreased in the third-instar nymphs injected with double-stranded RNA (dsRNA). The survival rate of different TRP dsRNA injected nymphs all exceeded 81%, which was no significant difference compared with the control group. These results suggested that these 9 TRP channels are expressed throughout the body and all ages of the brown planthopper, and are involved in regulating multiple physiological and behavioral processes. The identification of TRP channel genes in this study not only provides a foundation for further exploring the potential roles of TRP channels, but also serves as targets to develop new insecticides for the control of agricultural pests.

Melatonin promotes adventitious root formation in apple by promoting the function of MdWOX11.

BACKGROUND: Melatonin (MT) is important for plant growth and development; however, it is not known whether MT is involved in apple adventitious root (AR) development. In this study, we treated Malus prunifolia (MP) at four different stages of AR development, and analyzed the level of the endogenous hormones MT, auxin (IAA), zeatin-riboside (ZR), abscisic acid (ABA), and gibberellins (GA1 + 3) in all four treatment groups and the untreated control group. The expression of MT, IAA biosynthesis, transport and signal transduction, the cell cycle, and root development related genes were quantified by RT-qPCR. The function of MdWOX11 was analyzed in transgenic apple plants. RESULTS: The promotion of AR development by MT was dependent on the stage of AR induction between 0 and 2 d in apple rootstocks. MT-treatment increased the level of IAA and crosstalk existed between MT and IAA during AR formation. The expression of MdWOX11 was induced by MT treatment and positively regulated AR formation in apple. Furthermore, transgenic lines that overexpressed MdWOX11 lines produced more ARs than 'GL3'. Phenotypic analysis indicated that MdWOX11 overexpression lines were more sensitive to exogenous MT treatment than 'GL3', suggesting that MdWOX11 regulates AR formation in response to MT in apple rootstock. CONCLUSIONS: MT promotes AR formation mainly during the AR induction stage by inducing IAA levels and upregulating MdWOX11.

MdMYB88 and MdMYB124 Enhance Drought Tolerance by Modulating Root Vessels and Cell Walls in Apple.

Water deficit is one of the main limiting factors in apple (Malus × domestica Borkh.) cultivation. Root architecture plays an important role in the drought tolerance of plants; however, research efforts to improve drought tolerance of apple trees have focused on aboveground targets. Due to the difficulties associated with visualization and data analysis, there is currently a poor understanding of the genetic players and molecular mechanisms involved in the root architecture of apple trees under drought conditions. We previously observed that MdMYB88 and its paralog MdMYB124 regulate apple tree root morphology. In this study, we found that MdMYB88 and MdMYB124 play important roles in maintaining root hydraulic conductivity under long-term drought conditions and therefore contribute toward adaptive drought tolerance. Further investigation revealed that MdMYB88 and MdMYB124 regulate root xylem development by directly binding MdVND6 and MdMYB46 promoters and thus influence expression of their target genes under drought conditions. In addition, MdMYB88 and MdMYB124 were shown to regulate the deposition of cellulose and lignin root cell walls in response to drought. Taken together, our results provide novel insights into the importance of MdMYB88 and MdMYB124 in root architecture, root xylem development, and secondary cell wall deposition in response to drought in apple trees.

Pymetrozine activates TRPV channels of brown planthopper Nilaparvata lugens.

The commercial insecticide pymetrozine has been extensively used for brown planthopper control in East Asia. The transient receptor potential vanilloid (TRPV) channel, which consists of two proteins, Nanchung (Nan) and Inactive (Iav), has recently been shown to be the molecular target of pymetrozine in the fruit fly (Drosophila melanogaster) and pea aphid (Acyrthosiphon pisum). In this study, we characterized the Nan and Iav TRPV channel subunits of N. lugens and measured the action of pymetrozine on them. NlNan and NlIav are structurally similar to homologs from other insects. The expression pattern analysis of various body parts showed that NlNan and NlIav were both more abundantly expressed in antennae. When NlNan and NlIav were co-expressed in Xenopus laevis oocytes, they formed channels with high sensitivity to pymetrozine (EC50 = 5.5 × 10-8 M). Behavioral observation revealed that the gravitaxis defect in the fruit fly nan36a mutant was rescued by ectopically expressed NlNan and the rescued behavior could be abolished by pymetrozine. Our results confirm that NlNan and NlIav co-expressed complexes can be activated by pymetrozine both in vitro and in vivo and provide useful information for future resistance mechanism studies.

An atypical R2R3 MYB transcription factor increases cold hardiness by CBF-dependent and CBF-independent pathways in apple.

Apple (Malus × domestica) trees are vulnerable to freezing temperatures. However, there has been only limited success in developing cold-hardy cultivars. This lack of progress is due at least partly to lack of understanding of the molecular mechanisms of freezing tolerance in apple. In this study, we evaluated the potential roles for two R2R3 MYB transcription factors (TFs), MYB88 and the paralogous FLP (MYB124), in cold stress in apple and Arabidopsis. We found that MYB88 and MYB124 positively regulate freezing tolerance and cold-responsive gene expression in both apple and Arabidopsis. Chromatin-Immunoprecipitation-qPCR and electrophoretic mobility shift assays showed that MdMYB88/MdMYB124 act as direct regulators of the COLD SHOCK DOMAIN PROTEIN 3 (MdCSP3) and CIRCADIAN CLOCK ASSOCIATED 1 (MdCCA1) genes. Dual luciferase reporter assay indicated that MdCCA1 but not MdCSP3 activated the expression of MdCBF3 under cold stress. Moreover, MdMYB88 and MdMYB124 promoted anthocyanin accumulation and H2 O2 detoxification in response to cold. Taken together, our results suggest that MdMYB88 and MdMYB124 positively regulate cold hardiness and cold-responsive gene expression under cold stress by C-REPEAT BINDING FACTOR (CBF)-dependent and CBF-independent pathways.

Knockdown of the ionotropic γ-aminobutyric acid receptor (GABAR) RDL gene decreases fipronil susceptibility of the small brown planthopper, Laodelphax striatellus (Hemiptera: Delphacidae).

Insect γ-aminobutyric acid receptors (GABARs) are important molecular targets of cyclodiene and phenylpyrazole insecticides. Previously GABARs encoding rdl (resistant to dieldrin) genes responsible for dieldrin and fipronil resistance were identified in various economically important insect pests. In this study, we cloned the open reading frame cDNA sequence of rdl gene from fipronil-susceptible and fipronil-resistant strains of Laodelphax striatellus (Lsrdl). Sequence analysis confirmed the presence of a previously identified resistance-conferring mutation. Different alternative splicing variants of Lsrdl were noted. Injection of dsLsrdl reduced the mRNA abundance of Lsrdl by 27-82%, and greatly decreased fipronil-induced mortality of individuals from both susceptible and resistant strains. These data indicate that Lsrdl encodes a functional RDL subunit that mediates susceptibility to fipronil. Additionally, temporal and spatial expression analysis showed that Lsrdl was expressed at higher levels in eggs, fifth-instar nymphs, and female adults than in third-instar and fourth-instar nymphs. Lsrdl was predominantly expressed in the heads of 2-day-old female adults. All these results provide useful background knowledge for better understanding of fipronil resistance related ionotropic GABA receptor rdl gene expressed variants and potential functional differences in insects.

Comparison of Insecticide Susceptibilities of Empoasca vitis (Hemiptera: Cicadellidae) from Three Main Tea-Growing Regions in China.

Empoasca vitis (Göthe) is an important insect pest in tea-growing areas of China, and chemical control is the main tactic for the management of this pest. Due to the pressure of increasing insecticide resistance and more stringent food safety regulations, development of sound IPM strategies for E. vitis is an urgent matter. This study comparatively evaluated four field populations of E. vitis from three different tea-growing regions in China for their susceptibilities to eight insecticides using a simple leaf-dip methodology. E. vitis was found to be most sensitive to indoxacarb (LC50<0.5 mg/liter) and least sensitive to isoprocarb (LC50>5 mg/liter) and sophocarpidine (LC50>95 mg/liter, a botanical pesticide) regardless of populations. Population (geographical) variations were higher for indoxacarb and imidacloprid than other compounds. Judging by the 95% fiducial limits of LC50 values, all populations had similar susceptibilities to chlorfenapyr, bifenthrin, and acetamiprid or imidacloprid. Correlation analysis suggested that chlorfenapyr and indoxacarb or isoprocarb may have a high risk of cross resistance. Considering potency (LC50) and maximum residual levels, chlorfenapyr and bifenthrin are good insecticide options followed by acetamiprid and indoxacarb. These results provide valuable information to intelligently select insecticides for IPM programs that are efficacious against E. vitis while also managing insecticide resistance and maximum residual levels for tea production in China.

Apple MdZAT5 mediates root development under drought stress.

Root plays an important role in plant drought tolerance, especially in horticultural crops like apples. However, the crucial regulator and molecular mechanism in root development of apple trees under drought are not well unknown. Cys2/His2-type Zinc-finger proteins are essential for plant response to drought, while the members of C2H2 Zinc-finger proteins in apple are largely unknown. In this study, we identified the members of the C1-2i subclass family of C2H2 Zinc-finger proteins in apple (Malus × domestica). Among them, MdZAT5 is significantly induced in apple roots under drought conditions and positively regulates apple root development under drought. Further investigation revealed that MdZAT5 positively regulates root development and root hydraulic conductivity by mediating the transcription level of MdMYB88 under drought stress. Taken together, our results demonstrate the importance of MdZAT5 in root development under drought in apple trees. This finding provides a new candidate direction for apple breeding for drought resistance.

Interfering small ubiquitin modifiers (SUMO) improves the thermotolerance of apple by facilitating the activity of MdDREB2A.

Heat stress, which is caused by global warming, threatens crops yield and quality across the world. As a kind of post-translation modification, SUMOylation involves in plants heat stress response with a rapid and wide pattern. Here, we identified small ubiquitin modifiers (SUMO), which affect drought tolerance in apple, also participated in thermotolerance. Six isoforms of SUMOs located on six chromosomes in apple genome, and all the SUMOs were up-regulated in response to heat stress condition. The MdSUMO2 RNAi transgenic apple plants exhibited higher survival rate, lower ion leakage, higher catalase (CAT) activity, and Malondialdehyde (MDA) content under heat stress. MdDREB2A, the substrate of MdSUMO2 in apple, was accumulated in MdSUMO2 RNAi transgenic plants than the wild type GL-3 at the protein level in response to heat stress treatment. Further, the inhibited SUMOylation level of MdDREB2A in MdSUMO2 RNAi plants might repress its ubiquitination, too. The accumulated MdDREB2A in MdSUMO2 RNAi plants further induced heat-responsive genes expression to strengthen plants thermotolerance, including MdHSFA3, MdHSP26.5, MdHSP18.2, MdHSP70, MdCYP18-1 and MdTLP1. In summary, these findings illustrate that interfering small ubiquitin modifiers (SUMO) in apple improves plants thermotolerance, partly by facilitating the stability and activity of MdDREB2A.

Heterologous Overexpression of Apple MdKING1 Promotes Fruit Ripening in Tomato.

Fruit ripening is governed by a complex regulatory network, and ethylene plays an important role in this process. MdKING1 is a γ subunit of SNF1-related protein kinases (SnRKs), but the function was unclear. Here, we characterized the role of MdKING1 during fruit ripening, which can promote fruit ripening through the ethylene pathway. Our findings reveal that MdKING1 has higher expression in early-ripening cultivars than late-ripening during the early stage of apple fruit development, and its transcription level significantly increased during apple fruit ripening. Overexpression of MdKING1 (MdKING1 OE) in tomatoes could promote early ripening of fruits, with the increase in ethylene content and the loss of fruit firmness. Ethylene inhibitor treatment could delay the fruit ripening of both MdKING1 OE and WT fruits. However, MdKING1 OE fruits turned fruit ripe faster, with an increase in carotenoid content compared with WT. In addition, the expression of genes involved in ethylene biosynthesis (SlACO1, SlACS2, and SlACS4), carotenoid biosynthesis (SlPSY1 and SlGgpps2a), and fruit firmness regulation (SlPG2a, SlPL, and SlCEL2) was also increased in the fruits of MdKING1 OE plants. In conclusion, our results suggest that MdKING1 plays a key role in promoting tomato fruit ripening, thus providing a theoretical basis for apple fruit quality improvement by genetic engineering in the future.

Transcriptome analysis reveals the regulatory mechanism by which MdWOX11 suppresses adventitious shoot formation in apple.

Adventitious shoot (AS) regeneration accelerates plant reproduction and genetic transformation. WOX11 is involved in many biological processes, but its regulation of AS regeneration has not been reported. Here, we showed that the genotype and CK/IAA ratio of apple leaves were the key factors that affected their capacity for AS formation. Moreover, the expression level of MdWOX11 was negatively correlated with the capacity for AS formation. Phenotypic analysis of MdWOX11 transgenic plants showed that overexpression of MdWOX11 inhibited AS formation. Endogenous hormone analysis demonstrated that the contents of auxin (IAA), cytokinin (CK), and abscisic acid (ABA) were higher in MdWOX11-RNAi plants than in MdWOX11-OE transgenic plants. We used RNA sequencing to examine the transcriptional responses of genes in MdWOX11-RNAi and MdWOX11-OE transgenic apple plants at different AS stages. We identified 8066 differentially expressed genes and focused our analysis on those involved in the IAA, CK, ABA, and gibberellin (GA) hormone signaling pathways. The expression of genes related to the CK signaling pathway and shoot development was higher in GL-3 than in MdWOX11-OE transgenic plants during the callus and AS emergence stages. However, the expression of MdCKX5 was higher in MdWOX11-OE transgenic plants than in GL3 and MdWOX11-RNAi transgenic plants. Yeast one-hybrid (Y1H) assays, dual-luciferase reporter assays, and ChIP-qPCR showed that MdWOX11 binds to the promoter of MdCKX5, and a dual-luciferase reporter assay showed that MdWOX11 enhanced the promoter activity of MdCKX5. We concluded that MdCKX5 acts downstream of MdWOX11 to control AS formation, and we built a regulatory model of the suppression of AS formation by MdWOX11 in apple.